Smallpox is widely considered to be the most serious threat for use as a potential agent of biowarfare. There is an effective vaccine available (vaccinia virus), but because of a small but significant risk of serious complications (including death) associated with vaccinia virus, mass immunization is not currently advisable. This leaves the populace at risk to the deliberate or accidental introduction of a pathogenic poxvirus into our environment. The focus of this proposal is to develop an alternative vaccine with improved safety for use in preventing human disease caused by pathogenic orthopoxviruses such as variola virus. To accomplish this goal we will utilize our newly-developed BCV (bacterial commensal vector) technology. BCV utilizes gram-positive commensal bacteria, such as Streptococcus gordonii, to express heterologous antigens of interest on its external surface. Phase I human clinical trials indicate that this S. gordonii strain is safe and well-tolerated in humans. Mucosal immunization of mice with S. gordonii expressing either the vaccinia virus A27L protein or DSL elicited protection against lethal vaccinia challenge. We believe that the delivery of selected vaccinia virus antigens via this live bacterial vector system will provide an effective and safe method for prevention of smallpox in humans. The following specific aims are proposed 1) To construct recombinant S. gordonii strains that express conserved vaccinia antigens anchored to the bacterial surface, and 2) To test the immunogenicity of S. gordonir.W recombinants when delivered topically into mice, measuring antibody response (IgG and IgA), antigen-specific CTL responses, viral neutralization and/or killing, and protection against a live viral challenge in a murine model. Successful completion of the proposed [unreadable] experiments will identify a BCV smallpox vaccine candidate suitable for subsequent preclinical validation and development. [unreadable] [unreadable]